| Objective:Genetic factors are of vital importance in the pathogenesis of gout,while the genetic background of gout inflammatory arthritis response induced by sodium urate crystals is still largely unknown.Based on whole-genome sequencing analysis,we found a novel gout pathogenic gene PLAA in a primary gout family（35 patients,9 patients,4 generations）,but its pathogenesis is unclear.PLAA gene encodes phospholipase A2 activating protein,which is highly expressed in monocytes and macrophages and has previously been reported to involve in the induction of NF-κB signaling pathway.In this study,we used monosodium urate crystal（MSU）-treated human macrophages and PLAA monocyte-specific knockout mice as experimental models to focus on the role of PLAA gene in gout inflammation and its molecular mechanism.Methods:1.THP-1-derived macrophages were transfected with lentivirus to construct stable PLAA knockdown and overexpression models.Treated with MSU crystals to further clarify the molecular mechanism of PLAA involvement in gout inflammation.1.1 RT-q PCR and Western Blot were used to identify PLAA knockdown and overexpression efficiency.The transcript level of IL-1βand the protein level of pro-IL-β,and the secretion of IL-1βwas detected by Western Blot and ELISA to observe the effect of PLAA on the key inflammatory factor IL-1β.1.2 To explore the effect of PLAA on MSU-induced NF-κB activation by detecting the total protein and phosphorylation level of IκBαand the phosphorylation level of NF-kB（P65）by Western Blot.1.3.After overexpression of PLAA,the transcript levels of IKKα/βand its upstream signaling pathway were detected by RT-PCR.1.4.To detect changes in NLRP3 inflammasome components by RT-PCR and Western Blot after overexpression of PLAA.1.5.After knockdown/overexpression of PLAA,the transcript levels of IκBαwere detected by RT-PCR;the endogenous interactions of PLAA with VCP,IκBα,and NLRP3 were explored by immunoprecipitation to preliminarily investigate the regulatory mechanism of PLAA on NF-κB.2.Construction of PLAA monocyte-specific knockout mouse model.2.1.Bone marrow derived macrophages（BMDMs）from CTR mice and Plaa CKO mice were isolated,cultured with L929 cell supernatants for 7 days to induce them into macrophages, and PLAA knockdown efficiency was detected by Western Blot;after LPS pre-stimulation, MSU crystals were given for 6 hours,PLAA knockdown efficiency,pro-IL-βprotein levels and the transcription and secretion of IL-1βwere detected by RT-PCR and Western Blot.2.2.To confirm the mechanism of PLAA regulation of gouty inflammation,Western Blot was used to determine the expression of critical proteins in the NF-B pathway and the NLRP3 inflammasome in BMDMs.2.3.To establish a mouse gouty arthritis model by injecting MSU suspension into the foot pads,the thickness of the foot pads and the mechanical pain domain of mice were measured at 0,4,8,12,24,48 and 72 h after injection to investigate the effect of PLAA deficiency on gouty inflammation under physiological conditions.2.4.MSU suspension was injected into the foot pads of mice,and the soft tissues of the foot pads of both groups were collected 8 h after injection,and the levels of IκBα,NLRP3protein were detected by Western Blot.Results.1.In THP-1 cell line,RT-PCR and Western Blot results showed that MSU-induced IL-1β expression was downregulated with PLAA knockdown;overexpression of PLAA reversed IL-1βexpression.In addition,the phosphorylation levels of both IκBαand P65 were significantly increased after MSU induction in PLAA overexpressing cell lines compared to the null virus cell lines,while the total IκBαprotein levels were decreased,while the opposite trend was observed for NF-κB activation-related proteins after knockdown of PLAA.It was further found that mRNA and protein levels of NLRP3 were increased after overexpression of PLAA.ASC,pro-Caspase-1 protein levels did not change significantly,but Caspase-1 mature secretion in culture supernatant was significantly increased.2.After MSU stimulation in BMDMs,it was found that Plaa deficiency resulted in a significant decrease in IL-1βmRNA,pro-il-1βprotein levels,and IL-1βin cell supernatants,and diminished NF-κB activation,accompanied by an increase in total IκBαprotein levels and a decrease in NLRP3 protein.3.The footpad swelling peaked around 8 hours in the acute gouty arthritis model in mice, according to the findings.The degree of footpad swelling and nociception in Plaa CKO mice were always lower than those in control mice.4.Transcript levels of IKK kinase and its upstream MyD88 and TRAF6 were found to be elevated after overexpression of PLAA in THP-1 cells,but no difference in transcript levels of IκBαafter either knockdown or overexpression of PLAA.Endogenous immunoprecipitation assays observed the presence of PLAA binding to VCP and IκBα.Conclusion:In this study,we observed that PLAA deficiency attenuates the inflammatory response in a mouse model.At the cellular level,PLAA enhances NF-κB activation leading to increased expression of NLRP3 and Pro-IL-1β,which in turn promotes NLRP3 inflammatory vesicle activation and ultimately leads to increased IL-1βsecretion. |
